Pref-1 (preadipocyte factor-1) is a plasma membrane glycoprotein containing six EGF-like repeats in the extracellular domain. Pref-1 is highly expressed in 3T3-L1 preadipocytes, but is not detectable in mature fat cells. Pref-1 inhibits adipocyte differentiation in that constitutive expression of Pref-1 inhibits and antisense Pref-1 expression enhances adipocyte differentiation in vitro. We found that Pref-1 extracellular domain is cleaved to generate a biologically active 50 kD soluble form. We also demonstrated Pref-1 inhibition of adipogenesis in vivo: Pref-1 knockout mice that we produced are obese, while ectopic overexpression of Pref-1 made the transgenic mice lean. We recently found that Pref-1 interacts with a major ECM component, fibronectin and that excess C-terminal fibronectin domain that interacts with Pref-1 alleviates the Pref-1 inhibition of adipogenesis. The goal of this research is to elucidate the anti-adipogenic function of Pref-1. Aim 1 is to further examine Pref-1 function in adipogenesis. Using our Pref-1 null mouse embryo fibroblasts (MEFs) that differentiate into adipocytes, we will examine the stage and the mode of Pref-1 inhibition in vitro. We will further examine Pref-1 function in vivo using our Pref-1 knockout mice and transgenic mice overexpressing soluble Pref-1 by subjecting them to nutritional and genetic pressures. We will also generate transgenic mice expressing the membrane form of Pref-1 to compare its function with the soluble form. Aim 2 is to examine mechanisms for Pref-1 inhibition of adipogenesis. Using Pref-1 null MEFs, we will examine downstream signaling of Pref-1, especially the MAPK pathway. We will examine the requirement of fibronectin for Pref-1 effect and characterize Pref-1/fibronectin interaction, their interacting domains, and the biological consequences. Aim 3 is to generate transgenic mice expressing eGFP using Pref-1 promoter/gene. We will define in vivo the region necessary for Pref-1 expression in preadipocytes but not in adipocytes. This will also aid us to direct expression of other genes to preadipocytes of adipose tissue. This research is directed toward understanding adipocyte differentiation, a critical process that contributes to the development of excess adipose mass and obesity. In the long nm, elucidating the mechanisms of Pref-1 function and regulation will allow us to begin to develop strategies to control adipogenesis by modifying Pref-1 action and by altering expression of preadipocyte genes, including Pref-1, that control adipogenesis.